The present invention relates in part to protein kinases. In particular, the invention concerns the identification of protein kinase proteins which are human orthologues of the drosophila WART gene (hWART).
The following description is provided to aid in understanding the invention, but is not admitted to describe or constitute prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and thereby regulate diverse cellular processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety of serine, threonine, or tyrosine amino acids in proteins.
Enzymes that mediate phosphorylation of cellular effectors generally fall into two classes. The first class consists of protein kinases which transfer a phosphate moiety from nucleotide triposphates to protein substrates. The second class consists of protein phosphatases which hydrolyze phosphate moieties from phosphoryl protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases are generally divided into two classes: receptor and non-receptor type proteins. Protein kinases may also be divided into three classes based upon the amino acids they act upon: (1) Some catalyze the addition or hydrolysis of phosphate on serine or threonine only; (2) some catalyze the addition or hydrolysis of phosphate on tyrosine only; and (3) some catalyze the addition or hydrolysis of phosphate on serine, threonine, and tyrosine.
Altered protein kinase activity has been associated with multiple abnormal cellular functions, including increased cell proliferation. Increased cell proliferation can result from at least two cellular events: (i) mutation, chromosome translocation, or gene amplification of proto-oncogenes (Bishop, Cell 64:235-248, 1991), or (ii) inactivation, loss by mutation, chromosomal loss, mitotic recombination, or gene conversion of tumor suppressor genes (Lasko et al., Ann Rev Genet 25:281-314).
A large number of potential tumor suppressor genes have been isolated from Drosophila melanogaster, a species of fruit fly. Watson et al., J. Cell Sci. 18:19-33 , 1994. Potential tumor suppressor genes are identified in this organism by first deleting, obstructing, or mutating a gene, and then detecting over-proliferative cell growth of specific tissues in dissected larvae and pupae. Xu et al., Development 121:1053-1063, 1995. This organism provides an ideal system for identifying potential tumor suppressor genes as it reproduces rapidly and its genome is readily manipulated by persons skilled in the art.
An example of a putative tumor suppressor gene, identified in Drosophila is the wts gene. Loss or inactivation of both copies of the wts gene results in the growth of tumors on the legs and wings of the flies. Bryant et al., Development 1993 Supplement: 239-249, 1993. The large size of these tumors suggests that the cells undergo more divisions than normal. Id. In addition, the rounded shape of the tumors suggests that the division of the mutant cells is not preferentially oriented. Id. These observations taken together with the increased thickness of the cuticles around the mutant cells suggest that the wts gene regulates cell adhesion, cell contact inhibition, and/or cell boundary recognition in Drosophila.
Several of the genes characterized as potential tumor suppressors in Drosophila are cloned. In particular, the wts gene contains a region that bears sequence similarity to the catalytic regions of mammalian non-receptor serine/threonine protein kinases. Watson, BioEssays 17:673-676, 1995. However, the human orthologues of the drosophila wts gene have not been reported.
The invention relates in part to novel human orthologues of the Drosophila wts gene (hWARTs). The Drosophila wts gene encodes a non-receptor serine/threonine kinase. The properties of the human orthologues are described herein. The present invention concerns polypeptides of hWART, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to the polypeptides, assays utilizing the polypeptides, and methods relating to all of the foregoing.
The term xe2x80x9corthologuexe2x80x9d as used herein, refers to a gene that is more c related, in terms of nucleic acid sequence, to another gene than a gene which is a homologue. In the context of this invention, xe2x80x9chomologousxe2x80x9d indicates that the nucleotide sequences of two genes and/or the sequences of the gene products (e.g., amino acid sequences) have significant similarity, and that the gene products perform a similar cellular function. Thus, two homologous genes may have sequences which have 50, 60, 70, 80, 90, or greater percent nucleotide sequence identity. By xe2x80x9cclosely relatedxe2x80x9d in the context of this invention, it is meant nucleic acid sequences that have greater than 90% identity.
The hWARTS genes encode proteins that are potential drug targets for controlling aberrant cell proliferation. Unlike their Drosophila ortholog, the hWARTS genes may not function as tumor suppressor genes. While their mRNA is absent from most normal cells they are abundantly expressed in many types of tumor cells. However, based on the high degree of sequence identity in the catalytic and non-catalytic regions between the hWART proteins and the Drosophila wts, it is likely that the hWART genes are involved in regulating cell adhesion, cell contact inhibition, and/or cell boundary recognition, and in regulation of signal transduction pathways related to cell proliferation.
Thus, in a first aspect, the invention features an isolated, enriched, or purified nucleic acid molecule encoding an hWART polypeptide.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid it is meant a polymer of 14, 17, 21 or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide sequence present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it and thus is meant to be distinguished from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that xe2x80x9cenrichedxe2x80x9d does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
The term xe2x80x9csignificantxe2x80x9d here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes the sequence from naturally occurring enrichment events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones can be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The term is also chosen to distinguish clones already in existence which may encode hWARTs but which have not been isolated from other clones in a library of clones. Thus, the term covers clones encoding hWART which are isolated from other non-hWART clones.
The term xe2x80x9cnucleic acid moleculexe2x80x9d describes a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
The term xe2x80x9ccDNA cloningxe2x80x9d refers to hybridizing a small nucleic acid molecule, a probe, to genomic cDNA. The probe hybridizes (binds) to complementary sequences of cDNA.
The term xe2x80x9ccomplementaryxe2x80x9d describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. Thus if a nucleic acid sequence contains the following sequence of bases, thymine, adenine, guanine and cytosine, a xe2x80x9ccomplementxe2x80x9d of this nucleic acid molecule would be a molecule containing adenine in the place of thymine, thymine in the place of adenine cytosine in the place of guanine, and guanine in the place of cytosine. Because the complement can contain a nucleic acid sequence that forms optimal interactions with the parent nucleic acid molecule, such a complement can bind with high affinity to its parent molecule.
The term xe2x80x9chybridizexe2x80x9d refers to a method of interacting a nucleic acid sequence with a DNA or RNA molecule in solution or on a solid support, such as nitrocellulose, nylon or some combination of these materials. If a nucleic acid sequence binds to the DNA or RNA molecule with high affinity, it is said to xe2x80x9chybridizexe2x80x9d to the DNA or RNA molecule. The strength of the interaction between the probing sequence and its target can be assessed by varying the stringency of the hybridization conditions. Under highly stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having one or two mismatches out of 20 contiguous nucleotides.
Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Examples of hybridization conditions are shown in the examples described herein. High stringent conditions may mean conditions that are at least as stringent as the following: hybridization in 50% formamide, 5xc3x97SSC, 50 mM NaH3PO4, pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5xc3x97Denhart solution at 42 C. overnight; washing with 2xc3x97SSC, 0.1% SDS at 45 C.; and washing with 0.2xc3x97SSC, 0.1% SDS at 45 C. Those skilled in the art will recognize how such conditions can be varied to vary specificity and selectivity.
cDNAs are molecules that may be reverse-transcribed from fragments of message RNA from a genomic source. These fragments form a cDNA library of nucleic acid molecules. cDNA libraries are constructed from natural sources such as mammalian blood, semen, or tissue.
The term xe2x80x9csubtractive hybridizationxe2x80x9d refers to a method similar to cDNA cloning except that cDNA prepared from mRNA in unstimulated cells is added to mRNA in stimulated or different types of cells. cDNA/mRNA can then be precipitated to enrich the mRNA specific to the stimulation signal or different cell type.
The term xe2x80x9chWART nucleic acid moleculexe2x80x9d as used herein refers to a nucleic acid molecule that encodes an hWART polypeptide. hWART nucleic acid molecules can be identified by hybridization procedures and cloning procedures as described herein.
An hWART polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence. In preferred embodiments, the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, a nucleic acid sequence that hybridizes to the nucleic acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, or a functional derivative (as defined below) of either of the foregoing. The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be mammalian (human) blood, semen, or tissue and the nucleic acid may be synthesized by the triester or other method or by using an automated DNA synthesizer.
The term xe2x80x9cmammalianxe2x80x9d refers to such organisms as mice, rats, rabbits, goats, more preferably monkeys and apes, and most preferably humans.
In other preferred embodiments, the nucleic acid molecule of the invention comprises a nucleotide sequence that (a) encodes a polypeptide having the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurring hWART polypeptide; (d) encodes an hWART polypeptide having the full length amino acid sequence of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, except that it lacks one or more of the following segments of amino acid residues: 12-45, 55-151, 236-377, 404-520, 555-559, 601-702, 691-998, 1011-1086 of SEQ ID NO:3, or 1-33, 43-139, 342-466, 467-480, 514-518, 974-1048 of SEQ ID NO:4; (e) is the complement of the nucleotide sequence of (d); (f) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 from amino acid residues 12-45, 55-151, 236-377, 404-520, 555-559, 601-702, 691-998, 1011-1086 of SEQ ID NO:3, or 1-33, 43-139, 342-466, 467-480, 514-518, 974-1048 of SEQ ID NO:4; (g) is the complement of the nucleotide sequence of (f); (h) encodes a polypeptide having the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, except that it lacks one or more of the domains selected from the group consisting of an N-terminal domain, a catalytic domain, and a C-terminal domain; or (i) is the complement of the nucleotide sequence of (h). The nucleic acid molecule of the invention is isolated, enriched, or purified from, preferably, a mammal, or most preferably from a human.
In yet other preferred embodiments, the nucleic acid is an isolated conserved or unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, or for the design of PCR probes to facilitate cloning of additional polypeptides.
By xe2x80x9cconserved nucleic acid regionsxe2x80x9d, it is meant regions present on two or more nucleic acids encoding an hWART polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acids encoding hWARTs polypeptides are provided in Abe, et al. J. Biol. Chem. 19:13361, 1992 (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 5 out of 20 contiguous nucleotides.
By xe2x80x9cunique nucleic acid regionxe2x80x9d it is meant a sequence present in a full length nucleic acid coding for an hWART polypeptide that is not present in a sequence coding for any other known naturally occurring polypeptide. Such regions preferably comprise 14, 17, 21 or more contiguous nucleotides present in the full length nucleic acid encoding an hWART polypeptide. In particular, a unique nucleic acid region is preferably of human origin.
In yet another aspect, the invention relates to a nucleic acid vector comprising a nucleic acid molecule encoding an hWART polypeptide and a promoter element effective to initiate transcription in a host cell.
The term xe2x80x9cnucleic acid vectorxe2x80x9d relates to a single or double stranded circular nucleic acid molecule that can be transfected or transformed into cells and replicate independently or within the host cell genome. A circular double stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that the restriction enzymes operate upon are readily available to those skilled in the art. A nucleic acid molecule of the invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
Many techniques are available to those skilled in the art to facilitate transformation or transfection of the expression construct into a prokaryotic or eukaryotic organism. The terms xe2x80x9ctransformationxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d refer to methods of inserting an expression construct into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the host cell outer membrane or wall permeable to nucleic acid molecules of interest.
The term xe2x80x9cpromoter elementxe2x80x9d describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, can facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. The promoter element precedes the 5xe2x80x2 end of the hWART nucleic acid molecule such that the latter is transcribed into mRNA. Host cell machinery then translates mRNA into a polypeptide.
Those skilled in the art would recognize that a nucleic acid vector can contain many other nucleic acid elements besides the promoter element and the hWART nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosomal binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, periplasm or peroxisome localization signals, or signals useful for polypeptide purification.
The invention also features a nucleic acid probe for the detection of a nucleic acid encoding an hWART polypeptide in a sample.
The term xe2x80x9cnucleic acid probexe2x80x9d refers to a nucleic molecule that is complementary to and can bind a nucleic acid sequence encoding the amino acid sequence substantially similar to that set forth in SEQ ID NO:3, or SEQ ID NO:4.
In preferred embodiments, the nucleic acid probe hybridizes to nucleic acid molecules encoding at least 46 contiguous amino acids of the sequences set forth in SEQ ID NO:3, SEQ ID NO:4, or a functional derivative thereof. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under highly stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
Methods for using the probes include detecting the presence or amount of hWART RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to hWART RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for an hWART polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container having disposed therein a nucleic acid probe.
The invention also features a nucleic acid molecule as set forth in SEQ ID NO:l or SEQ ID NO:2 or fragments thereof, comprising one or more regions that encode an hWART polypeptide or an hWART domain polypeptide, where the hWART polypeptide or the hWART domain polypeptide is fused to a non-WART polypeptide. Such fused polypeptides include, for example, but are not limited to, a GST-fusion protein.
The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complimentary to an RNA sequence encoding an hWART polypeptide and a transcriptional termination region functional in a cell.
Another aspect of the invention relates to a recombinant cell or tissue comprising a nucleic acid molecule encoding an hWART polypeptide. The recombinant cell may comprise a nucleic acid molecule encoding either an hWART polypeptide; an hWART domain polypeptide; or an hWART polypeptide or hWART domain polypeptide fused to a non-WART polypeptide.
The term xe2x80x9crecombinant organismxe2x80x9d refers to an organism that has a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced to an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
The term xe2x80x9corganismxe2x80x9d relates to any living being comprised of a least one cell. An organism can be as simple as one eukaryotic cell or as complex as a mammal. Therefore, a recombinant organism can also be a recombinant cell.
The recombinant cell can be a eukaryotic or prokaryotic organism.
The term xe2x80x9ceukaryotexe2x80x9d refers to an organism comprised of cells that contain a nucleus. Eukaryotes are differentiated from xe2x80x9cprokaryotesxe2x80x9d which do not have a nucleus and lack other cellular structures found in eukaryotes, such as mitochondria and endoplasmic reticulum. Prokaryotes include unicellular organisms, such as bacteria while eukaryotes are represented by yeast, invertebrates, and vertebrates.
The recombinant cell can harbor a nucleic acid vector that is extragenomic. The term xe2x80x9cextragenomicxe2x80x9d refers to a nucleic acid vector which does not insert into the cell genome. Many nucleic acid vectors are designed with their own origins of replication allowing them to utilize the recombinant cell replication machinery to copy and propagate the vector nucleic acid sequence. These vectors are small enough that they are not likely to harbor nucleic acid sequences homologous to genomic sequences of the recombinant cell. Thus these vectors replicate independently of the host genome and do not recombine with or integrate into the genome.
A recombinant cell can harbor a portion of a nucleic acid vector in an intragenomic fashion. The term xe2x80x9cintragenomicxe2x80x9d defines a nucleic acid construct that is incorporated within the cell genome. Multiple nucleic acid vectors available to those skilled in the art contain nucleic acid sequences that are homologous to nucleic acid sequences in a particular organism""s genomic DNA. These homologous sequences will result in recombination events that integrate portions of the vector into the genomic DNA. Those skilled in the art can control which nucleic acid sequences of the vector are integrated into the cell genome by flanking the portion to be incorporated into the genome with homologous sequences in the vector.
Another aspect of the invention features an isolated, enriched, or purified hWART polypeptide.
By xe2x80x9chWART polypeptidexe2x80x9d it is meant an amino acid sequence substantially similar to the sequence shown in SEQ ID NO:3, SEQ ID NO:4, or fragments thereof. A sequence that is substantially similar will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of SEQ ID NO:3 or SEQ ID NO:4.
The hWART polypeptides of the present invention preferably have a substantially similar biological activity to the proteins encoded by the full length nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, or to the proteins with amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. By xe2x80x9cbiological activityxe2x80x9d it is meant an activity of the hWART protein in a cell. The biological activity of the hWART is related to some of the activities of the cell which include, but are not limited to, cell proliferation motogenesis, metastasis, tumor escape, cell adhesion, transformation, or apoptosis.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues in the two sequences by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of 6, 12, 18 or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide it is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that xe2x80x9cenrichedxe2x80x9d does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In another aspect, the invention features an isolated, enriched, or purified hWART polypeptide fragment.
By xe2x80x9chWART polypeptide fragmentxe2x80x9d it is meant an amino acid sequence that is less than the full-length amino acid sequence. The full-length amino acid sequences of hWART1 and hWART2 are shown in SEQ ID NO:3 and SEQ ID NO:4. Examples of fragments include hWART domains, hWART mutants and hWART-specific epitopes.
By xe2x80x9chWART domainxe2x80x9d it is meant a portion of the hWART polypeptide having homology to amino acid sequences from one or more known proteins wherein the sequence predicts some common function, interaction or activity. Well known examples of domains are the SH2 (Src Homology 2) domain (Sadowski, et al, Mol. Cell. Biol. 6:4396, 1986; Pawson and Schlessinger, Curr. Biol. 3:434, 1993), the SH3 domain (Mayer, et al, Nature 332:272, 1988; Pawson and Schlessinger, Curr. Biol. 3:434, 1993), and pleckstrin (PH) domain (Ponting, TIBS 21:245, 1996; Haslam, et al, Nature 363:309, 1993), all of which are domains that mediate protein:protein interaction or protein:lipid interaction, and the kinase catalytic domain (Hanks and Hunter, FASEB J 9:576-595, 1995). Computer programs designed to detect such homologies are well known in the art. The relative homology is at least 20%, more preferably at least 30% and most preferably at least 35%.
By xe2x80x9chWART mutantxe2x80x9d it is meant an hWART polypeptide which differs from the native sequence in that one or more amino acids have been changed, added or deleted. Changes in amino acids may be conservative or non-conservative. By xe2x80x9cconservativexe2x80x9d it is meant the substitution of an amino acid for one with similar properties such as charge, hydrophobicity, structure, etc. Examples of polypeptides encompassed by this term include, but are not limited to, (1) chimeric proteins which comprise a portion of an hWART polypeptide sequence fused to a non-hWART polypeptide sequence, for example, a polypeptide sequence of hemmaglutinin (HA), (2) hWART proteins lacking a specific domain, for example the catalytic domain, and (3) hWART proteins having a point mutation. An hWART mutant will retain some useful function such as, for example, binding to a natural binding partner, catalytic activity, or the ability to bind to an hWART specific antibody (as defined below).
By xe2x80x9chWART-specific epitopexe2x80x9d it is meant a sequence of amino acids that is both antigenic and unique to an hWART polypeptide. An hWART-specific epitope can be used to produce hWART-specific antibodies, as more fully described herein. Particularly preferred epitopes are shown in the Examples section below.
By xe2x80x9crecombinant hWART polypeptidexe2x80x9d it is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
The polypeptide of the invention comprises an amino acid sequence having (a) the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4; (b) the full length amino acid sequence of the sequence set forth in SEQ ID NO:3 or SEQ ID NO:4, except that it lacks one or more of the following segments of amino acid residues:12-45, 55-151, 236-377, 404-520, 555-559, 601-702, 691-998, 1011-1086 of SEQ ID NO:3, or 1-33, 43-139, 342-466, 467-480, 514-518, 974-1048 of SEQ ID NO:4; (c) the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 from amino acid residues 12-45, 55-151, 236-377, 404-520, 555-559, 601-702, 691-998, 1011-1086 of SEQ ID NO:3, or 1-33, 43-139, 342-466, 467-480, 514-518, 974-1048 of SEQ ID NO:4; or (d) the full length amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4 except that it lacks one or more of the domains selected from the group consisting of an N-terminal domain, a catalytic domain, and a C-terminal domain.
In yet another aspect, the invention features an antibody (e.g., a monoclonal or polyclonal antibody), or antibody fragment, having specific binding affinity to an hWART polypeptide or hWART polypeptide fragment.
By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to target (hWART) polypeptides with greater affinity than it binds to other polypeptides under specified conditions. Antibodies having specific binding affinity to an hWART polypeptide may be used in methods for detecting the presence and/or amount of an hWART polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the hWART polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
The term xe2x80x9cpolyclonalxe2x80x9d refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof. For the production of polyclonal antibodies, various host animals may be immunized by injection with the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species.
xe2x80x9cMonoclonal antibodiesxe2x80x9d are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art. See, for example, Kohler, et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
In another aspect, the invention features a hybridoma which produces an antibody having specific binding affinity to an hWART polypeptide. By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line which is capable of secreting an antibody, for example an hWART antibody. In preferred embodiments the hWART antibody comprises a sequence of amino acids that is able to specifically bind an hWART polypeptide.
The invention features a method for identifying human cells containing an hWART polypeptide, or a related sequence. The method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying hWART polypeptides (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
The invention also features methods of screening cells for natural binding partners of hWART polypeptides. By xe2x80x9cnatural binding partnerxe2x80x9d it is meant a protein that interacts with an hWART polypeptide. Binding partners include ligands, agonists, antagonists and downstream signaling molecules such as adaptor proteins and may be identified by techniques well known in the art such as co-immunoprecipitation or by using, for example, a two-hybrid screen. (Fields and Song, U.S. Pat. No. 5,283,173, issued Feb. 1, 1994 and, incorporated be reference herein.) The present invention also features the purified, isolated or enriched versions of the polypeptides identified by the methods described above.
In another aspect, the invention provides a method for identifying a substance capable of modulating hWART activity comprising the steps of (a) contacting an hWART polypeptide with a test substance; and (b) determining whether the substance alters the activity of said polypeptide.
The invention also features another method of identifying substances capable of modulating the function of an hWART polypeptide. The method comprises the following steps: (a) expressing an hWART polypeptide in cells; (b) adding a compound to the cells; and (c) monitoring a change or an absence of a change in cell phenotype, cell proliferation, catalytic activity of the hWART polypeptide, and binding a natural binding partner.
The term xe2x80x9ccompoundxe2x80x9d includes small organic molecules including, but not limited to, oxindolinones, quinazolines, tyrphostins, quinoxalines, and those contained within extracts from natural sources. Examples of such compounds are included in section XIII, below.
The term xe2x80x9cfunctionxe2x80x9d refers to the cellular role of a serine-threonine protein kinase. The serine-threonine protein kinase family includes members that regulate many steps in signaling cascades, including cascades controlling cell growth, migration, differentiation, gene expression, muscle contraction, glucose metabolism, cellular protein synthesis, and regulation of the cell cycle.
The term xe2x80x9cmodulatesxe2x80x9d refers to the ability of a compound to alter the function of a protein kinase. A modulator preferably activates the catalytic activity of a protein kinase, more preferably activates or inhibits the catalytic activity of a protein kinase depending on the concentration of the compound exposed to the protein kinase, or most preferably inhibits the catalytic activity of a protein kinase.
The term xe2x80x9ccatalytic activityxe2x80x9d, in the context of the invention, defines the ability of a protein kinase to phosphorylate a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a product as a function of time. Phosphorylation of a substrate occurs at the active-site of a protein kinase. The active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
The term xe2x80x9csubstratexe2x80x9d as used herein refers to a molecule that is phoshorylated by or directly interacts with the protein kinase. The substrate is preferably a peptide and more preferably a protein. In relation to the protein kinase RAF, preferred substrates are MEK and the MEK substrate MAPK.
The term xe2x80x9cactivatesxe2x80x9d refers to increasing the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner or catalytic activity.
The term xe2x80x9cinhibitxe2x80x9d refers to decreasing the cellular function of a protein kinase. The protein kinase function is preferably the interaction with a natural binding partner or catalytic activity.
The term xe2x80x9cmodulatesxe2x80x9d also refers to altering the function of a protein kinase by increasing or decreasing the probability that a complex forms between a protein kinase and a natural binding partner. A modulator preferably increases the probability that such a complex forms between the protein kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the protein kinase and the natural binding partner depending on the concentration of the compound exposed to the protein kinase, and most preferably decreases the probability that a complex forms between the protein kinase and the natural binding partner.
The term xe2x80x9ccomplexxe2x80x9d refers to an assembly of at least two molecules bound to one another. Signal transduction complexes often contain at least two protein molecules bound to one another, either transiently or in succession. For instance, a receptor protein tyrosine kinase, GRB2, SOS, and RAF sequentially interact in response to a mitogenic ligand.
The term xe2x80x9cexpressingxe2x80x9d as used herein refers to the production of an hWART polypeptide from a nucleic acid vector containing an hWART gene within a cell. The nucleic acid vector is transfected into cells using well known techniques in the art as described herein.
The term xe2x80x9caddingxe2x80x9d as used herein refers to administering a solution comprising a compound to the medium bathing cells. The solution comprising the compound can also comprise an agent, such as dimethyl sulfoxide, which facilitates the uptake of the compound into the cells.
The term xe2x80x9cmonitoringxe2x80x9d refers to observing the effect of adding the compound to the cells of the method. The effect can be manifested in a change in cell phenotype, cell proliferation, protein kinase catalytic activity, or in the interaction between a protein kinase and a natural binding partner.
The term xe2x80x9ccell phenotypexe2x80x9d refers to the outward appearance of a cell or tissue or the function of the cell or tissue. Examples of cell or tissue phenotype are cell size (reduction or enlargement), cell proliferation (increased or decreased numbers of cells), cell differentiation (a change or absence of a change in cell shape), cell survival, apoptosis (cell death), or the utilization of a metabolic nutrient (e.g., glucose uptake). Changes or the absence of changes in cell phenotype are readily measured by techniques known in the art.
The term xe2x80x9ccell proliferationxe2x80x9d refers to the rate at which a group of cells divides. The number of cells growing in a vessel can be quantitated by a person skilled in the art when that person visually counts the number of cells in a defined area using a comon light microscope. Alternatively, cell proliferation rates can be quantitated by laboratory apparatae that optically measure the density of cells in an appropriate medium.
The method can utilize any of the molecules disclosed in the invention. These molecules include nucleic acid molecules encoding hWART polypeptides, nucleic acid vectors, recombinant cells, polypeptides, or antibodies of the invention.
Substances identified as modulators of hWART activity can be used to study the effects of hWART modulation in animal models of cell proliferative disorders. For example, inhibitors of hWART activity can be tested as treatments for cell proliferative disorders such as leukemia or lymphoma using subcutaneous xenograph models in mice.
In a preferred embodiment, the invention provides a method for treating or preventing an abnormal condition by administering a compound which is a modulator of hWART function in vitro. The abnormal condition preferably involves abnormality in hWART signal transduction pathway, and most preferably is cancer. Such compounds preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (such as the assays described in example 9 below). Examples of substances that can be screened for favorable activity are provided in section XIII below.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description, and from the claims.